Anionic charge-dynamic polymers for release of cationic agents

ABSTRACT

Materials and methods for the generation of polyelectrolyte multilayers that can erode to release cationic components. The multilayers comprise layers that contain one or more cations and one or more charge-dynamic anionic polymers. Charge-dynamic anionic polymers contain side chains having removable functional groups. Removal of the functional groups results in a change in the net change in the charge of the polymer which can disrupt interactions between cations and the anionic polymers and facilitate release of cations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 61/059,194, filed Jun. 5, 2008, which is incorporated by referencein its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract numberAI048717 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

The present invention relates to dynamic charge state anionic polymersthat are useful for release of cationic molecules, polyelectrolytemultilayers fabricated from them and methods of using the polymers.

Methods for the layer-by-layer assembly of multilayered polyelectrolytefilms (or ‘polyelectrolyte multilayers’) provide nanometer-scale controlover the structures and compositions of thin films fabricated from abroad range of cationic and anionic polymers.¹⁻⁴ An evolvingunderstanding of the structures and properties of these multilayeredmaterials—and the ways that they can be manipulated at various lengthscales—has contributed to the design of functional thin films ofinterest in a growing number of fundamental and applied contexts.³⁻¹⁰

Owing to the polyvalent nature of the electrostatic interactions inpolyelectrolyte multilayers, these assemblies are frequently regarded tobe ‘stable’ in physiologically relevant environments, i.e., theassemblies often do not dissolve readily or undergo large changes inmacroscopic film properties when incubated in physiologicalmedia.^(3,5,7 9) This general stability confers several potentialpractical advantages, and has contributed significantly to thedevelopment of polyelectrolyte multilayers in biomedical andbiotechnological contexts.

The present invention relates to certain polyelectrolyte multilayers andmethods that can be used to disrupt electrostatic interactions in suchmultilayers and promote the controlled disassembly of the multilayers inaqueous media.^(8,10) Methods that provide such control facilitate thedevelopment of thin films and coatings that permit the release ofprecise and well-defined quantities of chemical or biological agentsfrom the surfaces of macroscopic, microscopic, or nanoscopic objects.The present invention provides a new approach to the design ofpolyelectrolyte multilayers that provides control over film erosion andthe release of cationic agents from film-coated surfaces.

Several past studies have reported that it is possible to designpolyelectrolyte multilayers that erode, degrade, or disassemble inaqueous environments by fabricating them using polyelectrolytes withfunctionality that can be cleaved or degraded, for example, eitherhydrolytically,¹¹⁻¹⁶ enzymatically,¹⁷⁻¹⁹ or reductively.^(20,21) Theincorporation of degradable polyelectrolytes permits tunable and/ortriggered control over film disassembly and can be used to design filmsthat provide control over the release of a variety of differentagents.¹¹⁻²¹ Additional details on these and other past approaches topromoting film disruption can be found in several recent reviews.^(8,10)The majority of past reports on the incorporation of degradable polymersinto multilayered films have focused on the use of degradable cationicpolymers and thus, in general, on films designed to erode and controlthe release anionic polyelectrolytes (e.g., DNA).^(8,10)

In principle, the fabrication of polyelectrolyte multilayers usingdegradable anionic polymers would provide a platform for the design ofthin films that permit control over the release of cationic agents(e.g., cationic proteins, peptides, polymers, nanoparticles, etc.).There have been reports that enzymatically degradable anionic polymerscan be used to fabricate multilayers that degrade in the presence ofspecific enzymes^(17,22) or upon contact with cells.^(17,23) Progresstoward the fabrication of films using hydrolytically degradable anionicpolymers, however, has been limited by the dearth of commerciallyavailable synthetic polymers that are both anionic and degradable, and,more generally, by the challenges associated with the synthesis of suchpolymers. Thus, there is a need in the art for synthetic polymers thatare anionic and degradable.

It has been reported²⁴⁻²⁹ recently that it is possible to design‘charge-shifting’ cationic polymers (that is, cationic polymers thatundergo dynamic reductions in their net charge, which can also be calledcharge-dynamic polymers) by designing polymers inter alia withamine-functionalized side chains that can be cleaved hydrolytically.Polymer 1 (Eq 1) presents an example of this approach;²⁴ this polymerundergoes a gradual reduction in net charge (e.g., from cationic toanionic) upon side chain hydrolysis. Polymer 1 can, for example, be usedto fabricate multilayers that release plasmid DNA (negatively chargedspecies) for up to three months.

Both film erosion and the release of DNA from mulitlayers containingpolymer 1 can be understood in terms of disruptions in the ionicinteractions in these assemblies that occur upon polymer side chainhydrolysis.²⁴ Published U.S. patent application 2005/0027064 publishedFeb. 3, 2005 relates to charge-dynamic polymers and delivery of anioniccompounds and provides additional examples of charge-dynamic polymerstructures and their applications.

SUMMARY OF THE INVENTION

The present invention provides materials and methods for the generationof multilayers that erode and release cationic film components. Thisapproach is based on the design of charge-dynamic anionic polymers.

The present invention provides dynamic charge state anionic polymers, ormore simply polymers, that include a polymeric backbone formed from oneor more different monomers. One or more removable functional group(s)is/are attached to one or more repeat units of the polymeric backbone.The dynamic charge state anionic polymer has an anionic charge densitywhich is a characteristic of the polymeric backbone and the functionalgroup or groups attached to the polymeric backbone. The net charge ofthe dynamic charge state anionic polymer increases when one or more ofthe removable functional group(s) is/are removed from the dynamic chargestate anionic polymer. The present polymers can also be part of acopolymer where one or more segments of the copolymer are the dynamiccharge state anionic polymer.

In specific embodiments, the polymer contains side chains havingremovable functional groups as defined herein, for example, amide sidechains. In specific embodiments, polymers that have amide side chainscan be synthesized from polymers having amine side chains, including,among others, poly(allyl amine), poly(vinyl amine), poly(ethyleneimine), poly(lysine), poly(amidoamine)dendrimers, and chitosan intowhich amide side chains can be introduced. The polymeric backbone can belinear, branched or hyperbranched.

In some embodiments, at least one of the one or more removablefunctional group(s) is a hydrolyzable group, such as a pendant amide.The one or more removable functional group(s) may also include a labilelinkage, such as an ester, an anhydride, an orthoester, a phosphoester,or an acetal. More specifically, the one or more removable functionalgroups are hydrolyzable under acidic conditions. The anionic polymer maycontain one or more different amide side groups or a mixture of sidegroups of various chemical structure wherein at least a portion (atleast 10 mol percent) of the side chains are amides.

In some embodiments, the polymer with amide side chains is a block of acopolymer containing two or more blocks and the other blocks havedifferent, polymer backbones. In some embodiments, the polymer of theinvention is a copolymer, including a block copolymer, comprisingportions with different amide side chains. In other embodiments, thepolymer of the invention is a copolymer, including a block copolymer,comprising two or more different repeat units with different polymerside chains of which at least one side chain is an amide side chain. Inspecific embodiments, the polymer of the invention is a copolymer,including a block copolymer, comprising two or more different repeatunits where the two or repeat units include those having different amideside chains which hydrolyze at different rates on contact with an acidicenvironment.

In some embodiments, the polymers of the invention are biodegradable andbiocompatible.

In the polymers of the present invention, the mole percent of the repeatunits comprising the polymeric backbone which are substituted with theone or more removable functional group(s) range from 10 to 100 percentor from about 10 percent to about 100 percent. In additionalembodiments, the mole percent of the repeat units substituted with aremovable functional group may range from about 30 percent to about 100percent, about 50 percent to about 100 percent or about 70 percent toabout 100 percent. In specific embodiments, the polymers of the presentinvention can on average have 5 or more repeating units. In additionalspecific embodiments, the polymers of the invention have 5 to severalhundred thousand repeating units. In more specific embodiments, polymersof this invention have from 5-500,000 repeating units, from 5-300,000repeating units, from 5-200,000 repeating units or from 5-100,000repeating units. The polymers of the present invention may have anydesired molecular weight, such as from 1,000 to 100,000 grams/mole, orfrom about 2,000 to 50,000 grams/mole.

The dynamic charge state anionic polymer can be associated with a ligandfacilitating the delivery of the polymer to a specific target, such as atarget cell.

The present polymers can be part of a copolymer, which can be composedof any other polymers, for example a polymer such as PEG or PEO, whichare commonly used to give stability toward protein adsorption. Thepresent polymer is generally anionic, but different functional groupsattached to the polymer can render the polymer zwitterionic. To impartan anionic charge to the polymer, the attached functional groups arenegatively charged. The present polymer may also be capable of bufferingchanges in pH which result from the make-up of the polymer backboneand/or the attached functional groups.

The present dynamic charge state anionic polymers may benon-immunogenic, non-toxic or both nonimmunogenic and non-toxic. In thepresent polymers, the polymeric backbone can be degradable ornondegradable. The present polymers do not require that any degradationof the backbone occur at the same time as the shift in anionic charge.

One skilled in the art will recognize that the measure of degradabilitywill be commensurate with the environmental conditions and desiredproperties for any particular application for the present polymers. Asone non-limiting example, for biomedical uses of the present polymers,the present invention contemplates polymers that degrade in a desiredtime frame (from an hour to a week to a month to a year) underphysiological conditions typically found in the body or in a cell orcell compartment (e.g., pH ranges from about 5.0 to 7.4, a temperatureof about 37° C.) and an ionic strength of a typical physiologicalsolution (generally around 130-150 mM NaCl, for example). In the presentinvention, the degradability of the polymer can be measured by a varietyof methods, including, but not limited to, GPC (gel permeationchromatography).

The present invention provides polyelectrolyte multilayers whichcomprise at least one layer which is formed from a dynamic charge stateanionic polymer of the invention or from a copolymer containing them.The polyelectrolyte multilayers comprise at least one and preferablymore than one layer formed from a dynamic charge state anionic polymerof this invention. The polyelectrolyte multilayers in addition maycontain layers formed from polymers other than dynamic charge stateanionic polymers. For example, the polyelectrolyte multilayers maycontain one or more layers formed from cationic polymers, anionicpolymers, neutral polymers or zwitterionic polymers in addition to oneor more layers formed from dynamic charge state anionic polymers. Thepolyelectrolyte multilayer may contain an agent that is intended to beselectively released from the polyelectrolyte multilayer. That agent maybe neutral, zwitterionic, anionic or cationic and in specificembodiments is cationic. The agent may be a therapeutic agent or adiagnostic agent and may be selected among others from protein, peptide,and small molecules.

In specific embodiments, polyelectrolyte multilayers of this inventioncomprise a plurality of cation/anionic polymer bilayers, particularlywherein one or more cations or other encapsulated species are to bereleased. In specific embodiments, bilayers containing an active agentto be released are optionally separated by one or more intermediatepolyelectrolyte bilayers which do not contain an active ingredient to bereleased. Each polyelectrolyte multilayer of the invention canoptionally comprise one or more top protective bilayers and/or one ormore base bilayers. One or more base bilayers can be formed, forexample, between a substrate surface and an cation/anionic polymerbilayer where the cation or cations therein are intended for controlledrelease. A plurality of such base layers may intervene between thesubstrate surface and any cation /anionic polymer bilayers. Base layers,if present, are the bottom most layers in a polyelectrolyte assembly(those closest to the substrate or surface). An intermediate bilayer ora plurality of intermediate layers may intervene between bilayers orpluralities of bilayers of cation/anionic polymers where the cation orcations are intended for controlled release. One or more top protectivebilayers can be positioned as the top most bilayers in a polyelectrolytemultilayer. Intermediate, top protective and base bilayers can comprisea anionic polymer of the invention and a cation (including a cationicpolymer) other than a cation, the release of which is intended to becontrolled. Intermediate, top protective and base bilayers can comprisea anionic or cationic polymer other than those specifically described inthe formulas herein, but which is degradable.

Polyelectrolyte multilayers can comprise a plurality of bilayerscontaining different cations or different anionic polymers. The layerscontaining the same or different cationic or anionic components can beordered in a variety of ways in the multilayer. For example, a pluralityof layers having the same cations and anionic polymers can be orderedsequentially in the multilayer. Alternatively, two or more layerscontaining different cationic or different anionic components can belayered sequentially in the multilayer.

The invention provides methods of forming such polyelectrolytemultilayers comprising a dynamic charge state anionic polymer. Theinvention further provides methods of disrupting such polyelectrolytemultilayers by selectively changing the net charge of the dynamic chargestate anionic polymer in the polyelectrolyte multilayer. The inventionadditionally provides methods for release of one or more agents,particularly therapeutic agents or diagnostic agents, from suchpolyelectrolyte multilayers by selective disruption of thepolyelectrolyte multilayer by selectively changing the net charge of thedynamic charge state anionic polymer. A selective change in the netcharge of the dynamic charge state anionic polymer can be obtained byselective removal of one or more anionic side chains of the dynamiccharge state anionic polymer.

The present invention also provides the present polymers complexed withone or more cationic agents thereby forming an interpolyelectrolytecomplex. Suitable cationic agents may be naturally-occurring, synthetic,or both. In some embodiments, suitable examples of cationic agentsinclude proteins and polypeptides. In other embodiments, the cationicmolecule or agent may be a therapeutic molecule or a diagnosticmolecule, and/or may be selected from a protein, peptide or smallmolecule.

The interpolyelectrolyte complex may have any desired size dependingupon the intended use of the interpolyelectrolyte complex. For example,when the interpolyelectrolyte complex is used for delivery of a cationicagent to a cell, the interpolyelectrolyte complex can be 50 nm to about400 nm, or from about 50 to about 250 nanometers, in size. In otherembodiments, the interpolyelectrolyte complex may be provided in alayered complex made up of one or more layers of the dynamic chargestate anionic polymer and one or more layers of the cationic agent.

Generally, the interpolyelectrolyte complex will be prepared by mixingthe dynamic charge state anionic polymer with the cationic agent,thereby allowing formation of the interpolyelectrolyte complex.

In some embodiments, the present polymer or interpolyelectrolyte complexmay be provided in a biologically compatible solution or a biologicalsolution. Further, the polymer may be provided with a pharmaceuticallyacceptable excipient or another completely different polymer (e.g.,another anionic polymer) which could be an “excipient” or could have anadded function. Accordingly, the present compounds includepharmaceutical compositions that include any of the polymers or mixturesdescribed herein.

The present invention also provides methods for controlled delivery orrelease of an agent which may be a therapeutic or diagnostic agent andin specific embodiments is a cationic agent. In specific embodiments,controlled delivery or release may be to a cell or tissue. Controlledrelease or delivery may be from a polyelectrolyte multilayer of thisinvention or from an interpolyelectrolyte complex of this invention.Release or delivery of the agent is controlled by selective change inthe net charge of a dynamic charge state anionic polymer as describedherein.

In the present methods, the target cell or tissue can be in vitro or invivo. Where the target cell or tissue is in vivo, the polyelectrolytemultilayer or the interpolyelectrolyte complex may be administered to amammal, including a human. Any suitable form of administration may beemployed. In some embodiments of the present methods, the tissue or cellis a eukaryotic cell. In specific embodiments, the tissue or cell isthat of a mammal, including a human.

In the present methods and polymers, removal of the one or more of theremovable functional group(s) from the dynamic charge state anionicpolymer may be at least partially hydrolytic, partially enzymatic and/orpartially photolytic removal. In a specific embodiment, removal of theone or more removable functional groups is not enzymatic. In a specificembodiment, removal of the one or more removable functional groups ishydrolytic. The present polymers and methods may also be designed sothat the removal of the one or more of the removable functional group(s)from the dynamic charge state anionic polymer occurs at a substantiallyconstant rate or does not occur at a constant rate.

The present invention also provides kits containing one or more polymersof this invention in combination with one or more of one or morecationic species for inclusion in polyelectrolyte multilayers anddelivery to a target cell or tissue, one or more substrates upon whichpolyelectrolyte multilayers may be formed; one or more media forfacilitating formation of polyelectrolyte multilayers; media and/orreagent for triggering hydrolytic, enzymatic or other removal of removalfunctional groups; or instructions for carrying out formation ofmultilayers and/or degradation of multilayers. Kits may include one ormore individual containers having a selected amount of the one or moreanionic charge dynamic polymers of the invention to carry out a desiredapplication for delivery of a desired cationic species. Kits may includeone or more individual containers having a selected amount of one ormore cationic species for inclusion in polyelectrolyte multilayers andfor release from such multilayers.

The invention also provides methods of preparing the polymers of theinvention and pharmaceutical compositions containing the polymers andone or more therapeutic, diagnostic, and/or prophylactic agent.

In yet another aspect of the invention, the polymers of this inventionare used to form nanometer-scale complexes with cationic agents.

In another aspect of the invention, the polymers are used to encapsulatetherapeutic, diagnostic, and/or prophylactic agents. In otherembodiments, the polymers of this invention are employed to coat (e.g.,to form one or more layers on) particles, including microparticles andnanoparticles. This coating of particles can include the formation ofpolyelectrolyte multilayers on microparticles or nanoparticles. Hollowmultilayer capsules can also be formed employing one or more dynamiccharge state anionic polymers of this invention. Microcapsules and/ornanocapsules can be formed by initial formation of a multilayer orpolyelectrolyte multilayer comprising a dynamic charge state anionicpolymer on a sacrificial particle, either microparticle or nanoparticle,which serves as a template. The sacrificial particle is thereafterdegraded, as is known in the art, leaving the microcapsule. The coatedmicroparticles and capsules can range from 1 micrometer to 500micrometers, for example. Coated nanoparticles are generally less than 1micron. In some such embodiments, the coated particles or capsules allowfor the delivery of small molecules, proteins, and/or peptides.Microparticles and nanoparticles may be prepared using any of thetechniques known in the art to make such particles, such as, forexample, double emulsion and spray drying. In some embodiments, coatedparticles and hollow capsules may be used for pH-triggered delivery ofthe encapsulated contents due to the pH-responsive nature of thepolymers.

In another aspect of the invention, the polymers are used to encapsulatea cationic agent which is to be delivered selectively in response to atrigger or delivered in a controlled manner over time. The cationicagent can for example be comprised in a multilayer film which alsocomprises one or more anionic charge dynamic polymers of this invention.In a specific embodiment, the cationic agent can be released from thefilm on contact with an environment of appropriate pH, e.g., acidic pH.The contact with the environment may be contact with an aqueous solutionof appropriate pH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Exemplary illustration of the method of this invention forcontrolled film erosion. Top: Hydrolysis of the citraconic amide sidechains of anionic ‘charge-shifting’ polymer 2 under acidic conditionsyields cationic poly(allylamine). Bottom: Polymer 2 (grey) is anionicand can be used to fabricate polyelectrolyte multilayers. Time-dependentchanges in the net charge of polymer 2 result in changes in the natureof the ionic interactions in the multilayers and promote film disruptionand the release of cationic film components (red). Polymer 2 and PAH areshown as being completely ionized for illustrative purposes (see text).

FIG. 2: Kinetics of side chain amide hydrolysis at 37° C. for polymer 2in deuterated phosphate buffer (500 mM, pH=7.4, ▪) and acetate buffer(500 mM, pH=5.0, ▴), respectively, as determined by 1H NMR spectroscopy.Data depicted with dashed lines correspond to the kinetics of theproximal (Δ) and distal (⋄) side chain isomers of polymer 2 at pH 5.0.

FIG. 3: Plot of ellipsometric thickness versus the number of PAH/polymer2 bilayers (▪, solid lines) or PAH/polymer 3 bilayers (▴, dashed lines)deposited on bare silicon substrates.

FIG. 4: Plot of film erosion versus time for multilayered filmsfabricated from fluorescently labelled PAH and polymer 2 (solid lines)incubated at 37° C. at pH 7.4 (▪) and pH 5.0 (□). This plot alsoincludes erosion profiles for films fabricated from polymer 3 (dashedlines) incubated at 37° C. at pH 7.4 (▴) and pH 5.0 (Δ). Filmthicknesses were determined using ellipsometry at each time point andare expressed as percentages of the original thicknesses of each film.

FIG. 5: Plot of coumarin fluorescence versus time showing the release ofcoumarin labeled PAH from multilayered films fabricated from usingpolymer 2 (solid lines) incubated at 37° C. at pH 7.4 (▪) and pH 5.0(□). This plot also includes release profiles for films fabricated frompolymer 3 (dashed lines) incubated at 37° C. at pH 7.4 (▴) and pH 5.0(Δ).

DETAILED DESCRIPTION OF THE INVENTION

The general approach of the present invention is based on the synthesisof anionic polymers that undergo dynamic changes in charge states (i.e.,from anionic to “less anionic” or to zwitterionic or cationic) totrigger the disruption of multilayers and/or interpolyelectrolytecomplexes and the release of agents (particularly therapeutic ordiagnostic agents), including among others, proteins, peptides, polymersor nanoparticles which may be cationic.

In one embodiment, the invention makes use of methods developed for thereversible conversion of amine functionality to carboxylic acidfunctionality by treatment with α-methyl derivatives of maleicanhydride.³⁰⁻³⁶ Past studies have demonstrated, for example, that theaddition of citraconic anhydride (10) to primary amines results in theformation of a citraconic amide, and that citraconic amides can behydrolyzed readily under acidic conditions to regenerate primary aminefunctionality (Eq 2).³⁰

While the hydrolysis of amide bonds in aqueous media generally occursvery slowly, the hydrolysis of citraconic amides (e.g., 15) occursrapidly under acidic conditions owing to intramolecular catalysisfacilitated by the neighboring carboxylic acid group, which ismaintained in close proximity to the amide bond by the conformationalrigidity of the nearby double bond.^(30,31) The kinetics and mechanismof this reaction have been investigated using small-molecule modelsystems,^(30,31) and this general approach has been applied to thechemical modification of proteins (e.g., by reaction withsurface-accessible lysine residues) as a means to reversibly manipulateprotein charge.³⁷⁻³⁹

Anionic polymers of the invention can be generated generally by thereaction of small-molecule anhydrides with amine side chains on apolymer backbone. In specific embodiments, anionic polymers of theinvention are prepared by the reaction of anhydrides of formulas I-IV,below, with primary amine side chains on a polymer backbone,

-   where n is 0 or 1 and where dotted lines indicate optional bonds, if    R₃ and R₄ are absent the bond in the ring is a double bond; and-   where R₁, R₂, and R₃ and R₄ (if present) are not particularly    limited except that these groups should not significantly interfere    with the ability of the anhydride to react with primary amines and    further that these groups should not contain cationic moieties which    decrease the anionic charge state of the polymer. In specific    embodiments, R₁, R₂, and R₃ and R₄ are selected from optionally    substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic    and heterocyclic groups. In addition, R₁ and R₂ can together form a    5-10 member carbocyclic or heterocyclic ring which may be aryl or    heteroaryl or which can contain one or two double bonds. X    represents substitution on the indicated ring with one to four    substituents selected from hydrogen, halogen, hydroxyl, alkyl,    alkenyl, alkynyl, aryl, heteroaryl, carbocyclic or heterocyclic    groups and two X substituents can together form a 5-10 member    carbocyclic or heterocyclic ring which may be aryl or heteroaryl or    which can contain one or two double bonds. R₁, R₂, and R₃ and R₄    groups and X substituents which are alkyl, alkenyl, alkynyl, aryl,    heteroaryl, carbocyclic or heterocyclic groups are optionally    substituted with one or more halogens, hydroxyl, —CN, alkoxyl, —COOH    (or —COO⁻), —COOR, or —CON(R′)₂, where R is alkyl, alkenyl, alkynyl,    aryl, heteroaryl, carbocyclic or heterocyclic and each R′ is    hydrogen or R.

In specific embodiments, R₃ and R₄ are absent and the indicated ringcontains a double bond. In further embodiments, R₁-R₄ are optionallysubstituted alkyl groups. In further embodiments, R₃ and R₄ are absent,the indicated ring contains a double bond and R₁ and R₂ are optionallysubstituted alkyl groups. In further embodiments, R₃ and R₄ are absent,the indicated ring contains a double bond, one of R₁ or R₂ is a hydrogenand the other of R₁ or R₂ is an optionally substituted alkyl group. In aspecific embodiment, X represents hydrogen substitutents on theindicated ring. In a specific embodiment, X represents one to fourhalogen substituents on the indicated ring. In a specific embodiment, nis 1. In another specific embodiment, n is 1, R₃ and R₄ are absent andthe indicated ring contains a double bond. In another specificembodiment, n is 1, R₃ and R₄ are absent, the indicated ring contains adouble bond and R₁ and R₂ are optionally substituted alkyl groups. Inspecific embodiments one or both of R₁ and R₂ are methyl groups.

In specific embodiments, the anionic polymers of this invention compriseamide side chains which carry a double bond as illustrated in formulasV, VI, VII and polymer 2. Reaction of the anhydride with a polymer orcopolymer having primary amine functionality results in the formation ofside chain amides and presents a straightforward and modular approach tothe generation of ‘charge-shifting’ anionic polymers, or anionicpolymers capable of undergoing changes in net charge (i.e., from netanionic to neutral or net cationic) upon side chain amide hydrolysis.

Several recent reports have demonstrated the application of thisapproach to the design of polymer-based carrier systems for the deliveryof nucleic acids,^(32,34,35) proteins,³³ or small molecules³⁶ thatcreate disruptive electrostatic interactions,^(33,36) unmask otherdisruptive functionality,^(32,34,35) or shed functionality that is nolonger needed³⁵ upon exposure to low pH environments (e.g., such asthose found in cell endosomes or lysosomes or other extracellularenvironments).

In specific embodiments, the invention provides anionic polymers offormula V:

where r is an integer ranging from 5 to 100,000, x and y are numbersrepresenting the mole percent of the indicated side chains in thepolymer where y represents from 10 to 100 mole percent of amide sidechain and x+y is 1. Additionally R₁-R₄, n and dotted lines are asdefined above and the wavy line represents a linker which covalentlyattaches the primary amine or the amide to the polymer backbone. Notealso that the polymer can contain one or more than one different amideside chains. The number y represents the total mole percent of amideside chains e.g., y1+y2+y3+y4 . . . +yn, where y1 to yn represent theindividual mole percent of different amide side chains. When there aremore than one different amide side chains the variables R₁-R₄ in thedifferent amides can be different, n may be different or the optionaldouble bond in the amide may be present or absent. In specificembodiments, all amide side chains in the polymer are the same. In otherspecific embodiments, there are two or more different amide side chains.In additional embodiments, there are two, three, four, five or sixdifferent amide side chains. In other specific embodiments, there aretwo different amide side chains which are stereoisomers of each other.In specific embodiments, y ranges from 0.1 to 0.3 (from 10-30 molepercent). In other embodiments, y ranges from 0.20 to 0.5 representing20 to 50 mole percent. In additional embodiments, y ranges from 25 to100 mole percent. In other embodiments, y is 100 mole percent.

The primary amine or amide is linked to the polymer backbone by a linkermoiety which in specific embodiments is an alkylene, or alkyleneoxylinker containing from 1 to 10 carbon atoms. In a specific embodiment,the linker is an alkylene linker. In a specific embodiment, the linkeris an alkylene linker of formula —(CH₂)_(m)—, where m is an integer thatranges from 1 to 10 and preferably ranges from 1 to 6 and in specificembodiments is 1, 2 or 3. In a specific embodiment, the linker is analkyleneoxy linker of formula —(CH₂)_(m1)—O—(CH₂)_(m2)—,—(CH₂)_(m1)—O—(CH₂)_(m2)—O—(CH₂)_(m3)—,—(CH₂)_(m1)—O—(CH₂)_(m2)—O—(CH₂)_(m3)—O—(CH₂)_(m4)—, or—(CH₂)_(m1)—O—(CH₂)_(m2)—O—(CH₂)_(m3)—O—(CH₂)_(m4)—O(CH₂)_(m5)—, wherem1, m2, and m3, m4, and m5, if present, are integers andm1+m2+m3+m4+m5=2-10. In more specific embodiments, m1 and m2 and m3, m4,and m5, if present, are the same; m1 and m2 and m3, m4, and m5, ifpresent are all 2 or all 3 or all 4; or m1 and m2 and m3, m4, and m5, ifnon-zero, are different.

The polymer of formula V is a generic formula for an initially anioniccharge dynamic polymer of this invention in which the charge can beshifted from anionic, to zwitterionic, to cationic, by hydrolysis ofamide groups. These anionic charge dynamic polymers can be used todestabilize polyelectrolyte multilayers and promote the release ofagents, particularly cationic agents, from surfaces coated with themultilayers in aqueous media. The hydrolysis can be triggered bydecreasing the pH of the medium to which the multilayers or the polymeris contacted.

In a more specific embodiment, the invention provides polymer VI andeven more specifically polymer 2.

where r, y1, y2, m and R₁ are as defined above. In a specific embodimentof formula VI, R₁ is selected from optionally substituted alkyl orphenyl groups. In more specific embodiments, R₁ is an unsubstitutedalkyl group having 1 to 6 carbon atoms or having 1 to 3 carbon atoms. Ina specific embodiment, y1 ranges from 0.1 to 0.9. In another embodiment,y1 ranges from 0.25 to 0.75. In another embodiment, yl ranges from 0.60to 0.80. In specific embodiments y1/y2 is 2 to 3. In specificembodiments, y1/y2 is 2.3.

In another embodiment, the invention provides polymer VII:

where r, m, and R₁ are as defined above and y1, y2 and y3 are numbersrepresenting the mole percent of the indicated side group where y1+y2+y3is 1; In a specific embodiment, R₁ is an optionally substituted C1-C6alkyl group. In another specific embodiment m is 1-6 or m is 1, 2 or 3.In specific embodiments y3 ranges from 0.01 to 0.90, y3 ranges from 0.25to 0.50 or y 3 ranges from 0.1 to 0.25. In specific embodiments y1/y2 is2 to 3. In specific embodiments, y1/y2 is 2.3.

In specific embodiments, the polymer of the above formulas is a block ofa block copolymer. In specific embodiments, the copolymer comprises oneor more blocks of PEG or PEO.

In specific embodiments, the polymers of the above formulas are employedto form multiple layer polyelectrolyte films. Each layer of such filmscomprises a bilayer comprising one or more cations and one or moreanionic polymers. In a specific embodiment, the one or more than one ofthe cations can be a cationic polymer. In a specific embodiment, abilayer can comprise a plurality of cations each carrying 1-4 positivecharges for each anionic polymer of this invention therein. In aspecific embodiment, a bilayer can comprise a polycation carrying aplurality of positive charges and an anionic polymer of this invention.In specific embodiments, a bilayer can comprise two or more differentcations or polycations. In specific embodiments, a bilayer can comprisetwo or more different charge dynamic anionic polymers of this invention.In specific embodiments, a multiple layer polyelectrolyte film canfurther comprise one or more layers that do not contain a charge dynamicanionic polymer. In specific embodiments, a multiple layerpolyelectrolyte film can further comprise one or more layers thatcontain a neutral, zwitterionic or cationic polymer which is not derivedby removal of groups from a dynamic anionic polymer.

Initial studies of the formation of multilayers employing an anioniccharge dynamic polymer used polymer 2 as described in the Examples.

The present polymers are dynamic charge state anionic polymers that haveanionic charge which is a characteristic of the polymeric backbone andthe functional groups attached to the polymeric backbone. Anionic chargemay be distributed throughout the polymer or in portions of the polymer,for example, in certain blocks of a block copolymer. Anionic charge maybe on the polymer backbone as well as in the functional groups on thepolymer, some or all of the anionic functional groups may be removable.The polymers are designed such that the net charge of the dynamic chargestate anionic polymer increases when one or more of the removablefunctional group(s), e.g., amides, is removed from the dynamic chargestate anionic polymer.

Based on these criteria, the polymer backbone is not particularlylimited, so long as a portion of the repeat units of the polymer haveremovable side chains that allow for charge shifting as defined herein,for example, amide side chains. Polymers of the invention can beprepared, for example, from a starting polymer in which a portion of therepeating units have primary amine side chains that can be converted toamides. In some embodiments, the polymer backbone is not charged.Generally speaking, the charge of the polymer is measured underconditions in which it will be used. In some cases the charge of thepolymer is measured at physiological pH. In general, suitable polymericbackbones for dynamic charge state anionic polymers of the invention areany natural or synthetic polymers or copolymers having primary aminegroups or which can be modified to incorporate primary amine groups.Specific examples of such suitable polymeric backbones include, amongothers, poly(allylamine), poly(vinylamine), branched poly(ethyleneimine), poly(lysine) or more generally polypeptides which containlysine, poly(amidoamine) dendrimers, polysaccharides having amine groupsor side groups with amine groups, such as chitosan. In the presentpolymers, the polymeric backbone may be linear, branched orhyperbranched. Note that generally, amide groups can be introduced intopolymers containing a primary amine group employing reaction withanhydrides as exemplified in equation 3 above.

The present polymer is generally anionic, but different functionalgroups attached to the polymer can render the polymer zwitterionic. Thepresent polymer may also be capable of buffering changes in pH whichresults from the make-up of the polymer backbone and/or the attachedfunctional groups.

Similar to the backbone, the identity of the one or more removablefunctional group(s) of the present polymers is not particularly limitedas long as removal of the one or more removable functional group(s)decreases the anionic charge density of the polymer. As used herein,“removable functional group” means a chemical group that, upon removal,will decrease the anionic charge density of the polymer. In a specificembodiment, the functional group that is removed is a group that is nota polymer which contains a plurality of repeating units. In a specificembodiment, the removable functional group is not PEG. The functionalgroup may, however, be an oligomer having 2 to 10 repeating units, suchas a disaccharide or oligosaccharide having 2-10 monosaccharides or anoligopeptide having 2 to 10 amino acids. As will be apparent to theskilled artisan, polymers whose anionic charge decreases (i.e., becomesless anionic) in this manner can have a variety of features. Forexample, the removable functional group may be negatively charged sothat removal of the removable functional group increases net charge.Additionally removal of the removable functional group may result ingeneration of a positively charged side chain further increasing netcharge. One example of such a scheme is provided when the removablegroup contains an anionic group linked to the polymer backbone via ahydrolyzable amide linkage. Other configurations that achieve the chargeshifting properties of the present polymers will be apparent to thoseskilled in the art. When removable functional groups provide apositively charged species after removal from the polymer backbone andthe backbone itself is neutral, then the present polymers can shift frombeing anionic to cationic when the removable functional group isremoved.

In the present polymers, the mole percent of the repeat units comprisingthe polymeric backbone substituted with the one or more removablefunctional group(s) ranges from about 10 percent to about 100 percent orfrom 10 percent to 100 percent. In additional embodiments, the molepercent of the repeat units attached to the one or more removablefunctional group may range from about 30 percent to about 100 percent,from about 50 percent to about 100 percent, or from about 70 percent toabout 100 percent. The polymers of the present invention may have anydesired molecular weight, such as from 1,000 to 100,000 grams/mole, orfrom about 2,000 to 50,000 grams/mole in some embodiments.

The present dynamic charge state anionic polymers can benon-immunogenic, non-toxic or both nonimmunogenic and non-toxic. In thepresent polymers, polymeric backbone can be degradable or nondegradable.In some embodiments, the polymers of the invention are biodegradable andbiocompatible.

The molecular weights of the polymers may range from 5,000 g/mol to over100,000 g/mol in some embodiments and from 4,000 g/mol to 50,000 g/molin other embodiments. In some embodiments, the polymers are relativelynon-cytotoxic. In other embodiments, the polymers are biocompatible andbiodegradable.

Synthesis of Polymers

The polymers of this invention may be prepared by any method known inthe art. In some embodiments, the polymers are prepared fromcommercially available starting materials. In other embodiments, thepolymers are prepared from easily and/or inexpensively prepared startingmaterials. Methods described herein can be employed to prepare anionicpolymers of this invention.

The synthesized polymer may be purified by any technique known in theart including, but not limited to, precipitation, crystallization,chromatography, etc. In some embodiments, the polymer need not bepurified. In some embodiments, the polymer is purified through repeatedprecipitations from an organic solvent (e.g., diethyl ether, hexane,etc.). In some embodiments, the polymer is isolated as a salt, such as ahydrochloride salt or a pharmaceutically acceptable salt. As would beappreciated by one of skill in this art, the molecular weight of thesynthesized polymer and the extent of cross-linking may be determined bythe reaction conditions (e.g., temperature, starting materials,concentration, order of addition, solvent, etc.) used in the synthesis(Odian Principles of Polymerization 3rd Ed., New York: John Wiley &Sons, 1991; Stevens Polymer Chemistry: An Introduction 2nd Ed., NewYork: Oxford University Press, 1990; each of which is incorporatedherein by reference).

In one embodiment, a library of different polymers is prepared inparallel. A different amount of the one or more removable functionalgroups is added to each vial in a set of vials used to prepare thelibrary. The array of vials is incubated at a temperature and length oftime sufficient to allow functionalization of the polymers to occur. Thepolymers may then be isolated and purified using techniques known in theart. The polymers may then be screened using high-throughput techniquesto identify polymers with a desired characteristic (e.g., solubility inwater, solubility at different pH, ability to bind small molecules,ability to form microparticles or nanoparticles, etc.).

Interpolyelectrolyte Complexes

The present invention also provides the present polymers complexed withone or more cations thereby forming an interpolyelectrolyte complex. Inthe interpolyelectrolyte complexes of the present invention, the cationbound by the polymer is not particularly limited. In some embodiments,the cation need only have a single positive charge. In specificembodiments, the cation is a cation other than a metal cation. Inspecific embodiments, the cation is an organic species having 6-50 or6-100 carbon atoms. In some embodiments, the cation need only have atleast two positive charges. Suitable examples of cationic moleculesinclude, among others, proteins, peptides, therapeutic molecules oragents, diagnostic molecules or agents, natural or synthetic polymers,prophylactic agents, small molecules, organometallic compounds, drugs,vaccines, immunological agents, and the like.

The cations may be naturally occurring or synthetic as syntheticpolycations may also be used to form interpolyelectrolyte complexes ofthe invention. In some embodiments, the polymers of the invention arecomplexed to a cationic molecule.

The present invention also provides polyelectrolyte multilayers whichcan be employed, for example, for controlled release or delivery ofagents, particularly therapeutic, diagnostic or prophylactic agents. Ingeneral any species that it is desired to be released or delivered canbe incorporated into polyelectrolyte multilayers. In specificembodiments, the agent is cationic. In other embodiments, the agent isan agent other than a cationic agent (e.g., anionic, neutral orzwitterionic). The agent can be selected from proteins, peptides,polysaccharides, saccharides, therapeutic molecules, diagnosticmolecules, prophylactic molecules, small molecules, natural polymers,synthetic polymers, organometallic compounds, drugs, vaccines,immunological agents and more specifically includes enzymes, structuralproteins, receptors, soluble receptors, ion channels, pharmaceuticallyactive proteins, cytokines, interleukins, antibodies, antibodyfragments, antigens, coagulation factors, albumin, growth factors,hormones, insulin, and the like. Polyelectrolyte multilayers of thisinvention are formed as is known in the art having at least one layer(i.e., polyelectrolyte bilayer) that is formed using a dynamic chargestate anionic polymer or copolymer of this invention.

Cationic agents or molecules can, among others, be small molecules,natural polymers or synthetic polymers. More specifically cationicagents can be proteins which may be enzymes, structural proteins,receptors, soluble receptors, ion channels, pharmaceutically activeproteins, cytokines, interleukins, antibodies, antibody fragments,antigens, coagulation factors, albumin, growth factors, hormones,insulin, etc.

Microparticles and Nanoparticles

The polymers of the present invention may also be used to form drugdelivery devices. The polymers may be used to encapsulate cationiccompounds including small molecules, proteins, peptides, metals,organometallic compounds, and the like. The polymers may be used toencapsulate a mixture of cationic species with neutral, zwitterionic oranionic species. Some of the present polymers possess one or moreproperties that make them particularly suitable in the preparation ofdrug delivery devices. Such properties may include 1) the ability of thepolymer to complex and protect labile agents; and 2) the ability toneutralize the charge on positively charged agents. In some embodiments,the polymers are used to coat (form one or more layers on) particlescontaining the agent to be delivered (the layers may also contain thesame or a different agent to be delivered.) In some such embodiments,the diameter of coated microparticles ranges from 500 nm to 50micrometers, from 1 micrometer to 20 micrometers, or from 1 micrometerto 10 micrometers. In other embodiments, the coated microparticles rangefrom 1-5 micrometers. In other embodiments, the coated particles areless than 1 micrometer (nanoparticles). The polymers of this inventioncan be employed to form microcapsules or nanocapsules which may containan agent to be delivered. The capsules are formed from polyelectrolytemultilayers in which are least one layer is formed from a dynamic chargestate anionic polymer or copolymer of this invention. Capsules can beformed, for example, by initial formation of polyelectrolyte multilayerson a particle and thereafter degrading the particle. Both micro- andnanoparticles can be formed. Methods for deposition of films ontoparticles, are well-known in the art⁵³.

The polymer of this invention may be combined with other polymers (e.g.,PEG, PLGA) to form the microspheres. Methods for the formation ofmicrospheres are well-known in the art. Microparticles may be preparedusing various methods. Examples of such methods include, but are notlimited to, spray drying, single and double emulsion solventevaporation, solvent extraction, phase separation, simple and complexcoacervation, and other methods well known to those of ordinary skill inthe art. In some embodiments, the methods for preparing the particlesare the double emulsion process and spray drying methods. The conditionsused in preparing the microparticles may be altered to yield particlesof a desired size or property (e.g., hydrophobicity, hydrophilicity,external morphology, “stickiness”, shape, etc.). The method of preparingthe particle and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may also depend on the agentbeing encapsulated and/or the composition of the polymer matrix.

Methods developed for making microparticles for delivery of encapsulatedagents are described in the literature (for example, please see Doubrow,M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRCPress, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release5:13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283, 1987;Mathiowitz et ai. J. Appl. Polymer Sci. 35:755-774, 1988; each of whichis incorporated herein by reference). Nanoparticles likewise can beformed by methods that are well-known in the art.

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, the particles can be sized, for example,using a sieve.

Polyelectrolyte multilayers may be formed, for example, as described inVazquez et al., J. Am. Chem. Soc. 124, 13992 (2002). The number oflayers in such multilayers is not particularly limited. Additionally,different layers of these multilayers can contain different polymersand/or anions. The present invention contemplates that these multilayerstructures can be used for controlled release of a desired agent ordelivery of multiple agents. As is understood by the skilled artisan,the film growth of the layered structure is primarily dictated byelectrostatic interactions, hydrophobic interactions, hydrogen bonding,salt concentration, and solution pH. The polyelectrolyte multilayers canalso be used to deliver cations to a selected environment, includingdelivery to a cell or tissue.

Agent

The agents to be delivered by the systems of the present invention maybe therapeutic, diagnostic, or prophylactic agents. Any cationicchemical compound to be administered to an individual may be deliveredusing the interpolyelectrolyte complex. The agent may be a smallmolecule, natural polymer, synthetic polymer, organometallic compound,protein, peptide, polynucleotide, metal, an isotopically labeledchemical compound, drug, vaccine, immunological agent, or the like. Anychemical compound to be administered to an individual or for whichcontrolled release or delivery is desired can be released or deliveredemploying polyelectrolyte multilayers of this invention.

In some embodiments, the agents are organic compounds withpharmaceutical activity. In another embodiment of the invention, theagent is a clinically used drug. In some such embodiments, the drug isan antibiotic, anti-viral agent, anesthetic, steroidal agent,anti-inflammatory agent, antineoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, or the like, or combinations thereof.

In some embodiments of the present invention, the agent to be deliveredor released may be a mixture of agents. For example, a local anestheticmay be delivered or released in combination with an anti-inflammatoryagent such as a steroid. Local anesthetics may also be administered withvasoactive agents such as epinephrine. As a further example, anantibiotic may be combined with an inhibitor of the enzyme commonlyproduced by bacteria to inactivate an antibiotic (e.g., penicillin andclavulanic acid). A neutral, zwitterionic or anionic species which maybe a therapeutic, diagnostic or prophylactic agent may be delivered orreleased from the multilayer along with a cationic species. In specificembodiments, the cationic species employed with the charge dynamicanionic polymer of this invention to form multilayers need not itself bethe agent that is to be delivered, a neutral zwitterionic or anionicspecies also encapsulated in the polyelectrolyte multilayer may be theagent that is to be released or delivered.

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium. Examples of materials useful for CAT and x-ray imaging includeiodine-based materials.

Prophylactic agents of the invention include, but are not limited to,antibiotics, nutritional supplements, and vaccines. Vaccines maycomprise isolated proteins or peptides, inactivated organisms andviruses, dead organisms and viruses, genetically altered organisms orviruses, and cell extracts. Prophylactic agents may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, alum, Freund's adjuvant, etc. Prophylactic agents includeantigens of such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponemapallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

Targeting Agents or Ligands

The polymers and multilayer structures (e.g., coated particles andcapsules) of the invention may be modified to include one or moretargeting agents or ligands, since it is often desirable to target aparticular cell, collection of cells, or tissue. A variety of targetingagents that direct pharmaceutical compositions to particular cells areknown in the art (see, for example, Cotten et al. Methods Enzym.217:618, 1993; incorporated herein by reference). The targeting agentsmay be included throughout the particle or may be only on the surface.The targeting agent may be a protein, peptide, carbohydrate,glycoprotein, lipid, small molecule, antibody, antibody fragment,receptor or the like. The targeting agent may be used to target specificcells or tissues or may be used to promote endocytosis or phagocytosisof the particle. Examples of targeting agents include, but are notlimited to, antibodies, fragments of antibodies, low-densitylipoproteins (LDLs), transferring, asialycoproteins, gp120 envelopeprotein of the human immunodeficiency virus (HIV), carbohydrates,receptor ligands, sialic acid, and the like. If the targeting agent isincluded throughout the structure, the targeting agent may be includedin the mixture that is used to form the particles. If the targetingagent is only present on the surface, the targeting agent may beassociated with (i.e., by covalent, hydrophobic, hydrogen boding, vander Waals, or other interactions) the formed particles using standardchemical techniques.

The amine groups on a given polymer can also be conjugated eitherdirectly to the amine groups or via spacer molecules, with targetingligands and the like. Preferably, only a portion of the available aminegroups are coupled to the ligand or spacer ligand such that the netcharge of the polymer is anionic. The target ligands conjugated to thepolymer direct the polymer-nucleic acid/drug complex to bind to specifictarget cells and penetrate into such cells (tumor cells, liver cells,heamatopoietic cells, and the like). The target ligands can also be anintracellular targeting element, enabling the transfer of the nucleicacid/drug to be guided towards certain favored cellular compartments(mitochondria, nucleus, and the like). In certain embodiments, theligands can be sugar moieties coupled to the amino groups. Such sugarmoieties are preferably mono- or oligo-saccharides, such as galactose,glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose,nytrose, triose, dextrose, trehalose, maltose, galactosamine,glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.

The conjugation of an acid derivative of a sugar with the polymer ispreferred in some embodiments. In some such embodiments of the presentinvention, lactobionic acid (4-0-˜-D-galactopyranosyl-D-gluconic acid)is coupled to the polymer. The galactosyl unit of lactose provides aconvenient targeting molecule for hepatocyte cells because of the highaffinity and avidity of the galactose receptor on these cells.

Other types of ligands that may be used include peptides such asantibodies or antibody fragments, cell receptors, growth factorreceptors, cytokine receptors, transferrin, epidermal growth factor(EGF), insulin, asialoorosomucoid, mannose-6-phosphate (monocytes),mannose (macrophage, some B cells), Lewisx and sialyl Lewisx(endothelial cells), N-acetyllactosamine (T cells), galactose (coloncarcinoma cells), and thrombomodulin (mouse lung endothelial cells),fusogenic agents such as polymixin Band hemaglutinin HAI,lysosomotrophic agents, nucleus localization signals (NLS) such asT-antigen, and the like.

Pharmaceutical Compositions

Once the polymer, interpolyelectrolyte complex, e.g. polymer complexedwith cationic molecule or compound) or multilayer structure (coatedmicorparticle or microcapsule) have been prepared, they may be combinedwith one or more pharmaceutical excipients to form a pharmaceuticalcomposition that is suitable to administer to animals. Animals includehumans as well as non-human animals, including, for example, mammals,birds, reptiles, amphibians, and fish. Preferably, the nonhuman animalis a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog,a cat, a primate, or a pig). An animal may be a transgenic animal. Aswould be appreciated by one of skill in this art, the excipients may bechosen based on the route of administration as described below, theagent being delivered, time course of delivery of the agent, or otherfactors.

Pharmaceutical compositions of the present invention and for use inaccordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose, and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; detergentssuch as Tween 80; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toanimals, orally, rectally, parenterally, intracisternally,intravaginally, intranasally, intraperitoneally, topically (as bypowders, creams, ointments, or drops), bucally, or as an oral or nasalspray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e.,microparticles, nanoparticles, polynucleotide/polymer complexes), theliquid dosage forms may contain inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension,or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may be used in the preparation of injectables.In some embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the microparticles ornanoparticles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of anpharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispersing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

The present invention also provides methods of administering the presentpolymers and complexes. Generally, these methods can involve contactingan interpolyelectrolyte complex of the present invention with one ormore cells, such as those that make up a tissue. In one embodiment, theinterpolyelectrolyte complex is administered to an animal. Theinterpolyelectrolyte complex can be administered in any suitable manner,such as in the manner and formulations described above. In someembodiments, in solution the side chain esters slowly hydrolyze,resulting in the release of the cationic component that is boundelectrostatically to the polymer.

The present invention also provides methods for delivering a cationiccompound to a cell or tissue. In one aspect the present methods involvecontacting a composition that includes a interpolyelectrolyte complex ofthe invention with a target cell thereby allowing the target cell touptake the composition. The polymer of the present invention is designedsuch that when the interpolyelectrolyte complex enters the target cell,one or more of the removable functional group(s) is removed from thedynamic charge state anionic polymer which increases the cationic chargedensity (or decreased the anionic charge density) of the dynamic chargestate anionic polymer. The decrease in the anionic charge of the polymeris caused by the removal of anionic charges which promotes dissociationof the interpolyelectrolyte complex into the dynamic charge stateanionic polymer and the cationic molecule, allowing for release of thecationic molecule or in particular the delivery of the cationic moleculeto the target cell or cell compartment, such as an endosome, cytosol ornucleus of the cell. In some methods, at least one of the one or more ofthe removable functional group(s) is removed from the dynamic chargestate anionic polymer in a nucleus, endosome or cytosol of the targetcell. In this manner, the interpolyelectrolyte complex can dissociateprimarily in the desired compartment of the target cell and deliver thecationic molecule to the target cell compartment. The present methodscan also involve providing the interpolyelectrolyte complex and/orpreparing the interpolyelectrolyte complex. Generally, theinterpolyelectrolyte complex will be prepared by mixing the dynamiccharge state anionic polymer with the cationic molecule thereby allowingformation of the interpolyelectrolyte complex.

In another aspect, the present methods involve bringing apolyelectrolyte multilayer film or coating into contact with anenvironment, including tissue or cells to which or into which an agentis to delivered or released. The multilayer includes at least one layer(i.e., a bilayer) comprising a charge dynamic anionic polymer of thisinvention and at least one cationic species and preferably comprises aplurality of such layers. The polymer of the present invention isdesigned such that when one or more of the removable functional group(s)is removed from the dynamic charge state anionic polymer the anioniccharge of the dynamic charge state anionic polymer decreases (or thecationic charge of the polymer increases). The decrease in the anioniccharge of the polymer is caused by the removal of anionic charges whichpromotes dissociation of polyelectrolyte layers in the multilayerallowing for release of the cationic species and/or a neutral,zwitterionic or anionic agent also comprised in the multilayer. Thepresent methods can also involve providing the polyelectrolytemultilayer. The environment into which the agent is released can be abiological environment, which for example can be human or animal tissue.

In the present methods, the target cell or tissue can be in vitro or invivo. Where the target cell or tissue is in vivo, theinterpolyelectrolyte complex can be administered to a mammal. In someembodiments of the present methods, the cell is a eukaryotic cell.

In the present methods and polymers, removal of the one or more of theremovable functional group(s) from the dynamic charge state anionicpolymer can be at least partially hydrolytic, partially enzymatic and/orpartially photolytic removal. The present polymers and methods can alsobe designed so that removal of the one or more of the removablefunctional groups from the dynamic charge state anionic polymer occursat a substantially constant rate or does not occur at a constant rate.Accordingly, in the present methods, the majority, or substantially all,of the cations can be delivered to a desired environment, which may be apart of the cell, such as the nucleus, endosome or cytosol.

The present invention also provides kits for carrying out the methodsdescribed herein. Kits generally contain one or more anionic chargedynamic polymers of the invention. In one embodiment, the kit comprisesinstructions for carrying out any of the methods described herein. Theinstructions can be provided in any intelligible form through a tangiblemedium, such as printed on paper, computer readable media, or the like.The present kits can also include one or more reagents, buffers, media,agents and/or disposable equipment in order to readily facilitateimplementation of the present methods. Examples of kit components can befound in the description above and in the following examples. Such kitsmay be used in hospitals, clinics, physician's offices or in patients'homes to facilitate the co-administration of the enhancing and targetagents. The kits may also include as an insert printed dosinginformation for the co-administration of the enhancing and targetagents.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

Definitions

The following are terms used in the present application:

The term “alkyl” as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, alkyl groups have from 1 to 12, from1 to 8 carbon atoms, from 1 to 6 or 1 to 3 carbon atoms. Examples ofalkyl radicals include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl,n-octyl, n-decyl, n-undecyl, and dodecyl. A “cycloalkyl” group is acyclic alkyl group typically containing from 3 to 8 ring members suchas, but not limited to, a cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, or cyclooctyl group.

The term “alkoxy” as used herein refers to an alkyl group, as previouslydefined, attached to the parent molecular moiety through an oxygen atom.Examples include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy groups.

The term “alkenyl” denotes a monovalent group derived from a hydrocarbonmoiety having at least one carbon-carbon double bond by the removal of asingle hydrogen atom. Alkenyl groups include, for example, ethenyl,propenyl, butenyl, I-methyl-2-buten-l-yl, and the like. Alkenyl groupsinclude those having from 2-12 carbon atoms, those having 2-8, and thosehaving 2-6 carbon atoms.

The term “alkynyl” as used herein refers to a monovalent group derivedform a hydrocarbon having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. Representative alkynyl groups includeethynyl, 2-propynyl (propargyl), I-propynyl, and the like. Alkynylgroups include those having from 2-12 carbon atoms, those having 2-8,and those having 2-6 carbon atoms.

The term “aryl” as used herein refers to carbocyclic ring systems havingat least one aromatic ring including, but not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, and indenyl groups, and the like.Aryl groups can beunsubstituted or substituted with substituentsselected from the group consisting of branched and unbranched alkyl,alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino,dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto,nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide. Inaddition, substituted aryl groups include tetrafluorophenyl andpentafluorophenyl.

The term “alkylene” refers to a diradical of a branched or unbranchedsaturated hydrocarbon chain, preferably having from 1 to 10 carbonatoms, including 1-6 carbon atoms, and 2-4 carbon atoms. This term isexemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),more generally —(CH₂)_(n)— where n is and integer from 1-about 20,including 1-10, 1-6 or 2, 3 or 4. Alkylene groups may be branched.Alkylene groups may be optionally substituted. Alkylene groups may haveup to two non-hydrogen substituents per carbon atoms which do notinterfere with removal of removable functional groups. Alkylene groupsare useful as linker groups herein.

The term “alkyleneoxy” refers to an alkylene group ads described abovein which one or more non-neighboring —CH—, —CH₂— or substituted —C— arereplaced with an oxygen atoms, e.g., —CH₂—CH₂—O—CH₂—CH₂—, —O—CH₂—CH₂—,—CHCH₃—O—CH₂—CH₂—. The alkyleneoxy group can be branched or unbranched.The carbons of alkyleneoxy groups are optionally substituted withnon-hydrogen substituents which do not interfere with removal ofremovable functional groups. Alkyleneoxy groups are useful as linkergroups herein.

The term carbocyclic is used generally herein to refer to groupscontaining one or more carbon rings. The groups may be aromatic or arylgroups. Rings may contain 3-10 carbon atoms and one, two or three doublebonds or a triple bond. These groups may include single rings of 3 to 8atoms in size and bi- and tri-cyclic ring systems which may includearomatic six-membered aryl or aromatic groups fused to a non-aromaticring.

The terms “heterocyclic” and “heterocyclyl”, are used broadly herein torefer to an aromatic, partially unsaturated or fully saturated 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms insize and bi- and tri-cyclic ring systems which may include aromaticsix-membered aryl or aromatic heterocyclic groups fused to anon-aromatic ring. These heterocyclic and heterocyclyl rings and groupsinclude those having from one to three heteroatoms independentlyselected from oxygen, sulfur, and nitrogen, in which the nitrogen andsulfur heteroatoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternary.

The terms “aromatic heterocyclic” or “heteroaryl” as used herein, referto a cyclic aromatic radical having from five to 12 ring atoms of whichone ring atom is selected from sulfur, oxygen, and nitrogen; zero, one,or two ring atoms are additional heteroatoms independently selected fromsulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon,the radical being joined to the rest of the molecule via any of the ringatoms. The term includes heteroaromatic rings fused to aryl ring or tocarbocylci rings. Examples of such aromatic heterocyclyl groups include,but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, and isoquinolinyl groups,and the like.

Specific heterocyclic and aromatic heterocyclic groups that may beincluded in the compounds of the invention include:3-methyl-4-(3-methylphenyl)piperazine, 3-methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine,4-(2-(bis-(2-propenyl)amino)ethyl)piperazine,4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine,4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine,4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine,4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine,4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine,4-(2-methylthiophenyl) piperazine, 4-(2-nitrophenyl)piperazine,4-(2nitrophenyl) piperazine, 4-(2-phenylethyl)piperazine, 4-(2pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine, 4-(2,3dimethylphenyl)piperazine, 4-(2,4-difluorophenyl) piperazine,4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl) piperazine,4-(2,5-dimethylphenyl)piperazine, 4-( 2,6-dimethylphenyl)piperazine,4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine,4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine,4-3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,4-( 3,5-dimethoxyphenyl)piperazine,4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine-,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

The term “hydrocarbon”, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. The hydrocarbon may be unsaturated, saturated, branched,unbranched, cyclic, polycyclic, or heterocyclic. Illustrativehydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl,methoxy, diethylamino, and the like. As would be known to one skilled inthis art, all valencies must be satisfied in making any substitutions.

The terms “substituted”, whether preceded by the term “optionally” ornot, and “substituent”, as used herein, refer to the ability, asappreciated by one skilled in this art, to change one functional groupfor another functional group provided that the valency of all atoms ismaintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents may also be further substituted (e.g., anaryl group substituent may be further substituted. For example, a nonlimiting example is an aryl group that may be further substituted with,for example, a fluorine group at one or more position.

When two entities are “associated with” one another as described herein,they are linked by a direct or indirect covalent or non-covalentinteraction. Preferably, the association is covalent. Desirablenon-covalent interactions include hydrogen bonding, van der Waalsinteractions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc.

As used herein, “biodegradable” compounds are those that, whenintroduced into cells, are broken down by the cellular machinery or byhydrolysis into components that the cells can either reuse or disposeof, in some cases without significant toxic effect on the cells (e.g.,fewer than about 20% of the cells are killed when the components areadded to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain embodiments,the chemical reactions relied upon to break down the biodegradablecompounds are uncatalyzed.

“labile bond” is a covalent bond that is capable of being selectivelybroken. That is, a labile bond may be broken in the presence of othercovalent bonds without the breakage of other covalent bonds. Forexample, a disulfide bond is capable of being broken in the presence ofthiols without cleavage of any other bonds, such as carbon-carbon,carbon-oxygen, carbon-sulfur, carbon-nitrogen bonds, which may also bepresent in the molecule. “Labile” also means cleavable.

A “labile linkage” is a chemical compound that contains a labile bondand provides a link or spacer between two other groups. The groups thatare linked may be chosen from compounds such as biologically activecompounds, membrane active compounds, compounds that inhibit membraneactivity, functional reactive groups, monomers, and cell targetingsignals. The spacer group may contain chemical moieties chosen from agroup that includes alkanes, alkenes, esters, ethers, glycerol, amide,saccharides, polysaccharides, and heteroatoms such as oxygen, sulfur, ornitrogen. The spacer may be electronically neutral, may bear a positiveor negative charge, or may bear both positive and negative charges withan overall charge of neutral, positive or negative.

In general, the “effective amount” of an active agent in a compositionor drug delivery device refers to the amount necessary to elicit thedesired biological response. As will be appreciated by those of ordinaryskill in this art, the effective amount of an agent or device may varydepending on such factors as the desired biological endpoint, the agentto be delivered, the composition of the encapsulating matrix, the targettissue, etc. For example, the effective amount of micro- ornanoparticles containing an antigen to be delivered to immunize anindividual is the amount that results in an immune response sufficientto prevent infection with an organism having the administered antigen.

As used herein, “peptide”, means peptides of any length and includesproteins. The terms “polypeptide” and “oligopeptide” are used hereinwithout any particular intended size limitation, unless a particularsize is otherwise stated. The only limitation to the peptide or proteindrug which may be utilized is one of functionality. The terms “protein”and “peptide” may be used interchangeably. Peptide may refer to anindividual peptide or a collection of peptides. peptides preferablycontain only natural amino acids, although non-natural amino acids(i.e., compounds that do not occur in nature but that can beincorporated into a polypeptide chain; see, for example,http://www.cco.caltech.edu/.about.da-dgrplUnnatstruct.gif, whichdisplays structures of non-natural amino acids that have beensuccessfully incorporated into functional ion channels) and/or aminoacid analogs as are known in the art may alternatively be employed.Also, one or more of the amino acids in an peptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. In some embodiments, the modifications of the peptidelead to a more stable peptide (e.g., greater half-life in vivo). Thesemodifications may include cyclization of the peptide, the incorporationof D-amino acids, etc. Typical of peptides that can be utilized arethose selected from the group consisting of oxytocin, vasopressin,adrenocorticotrophic hormone, epidermal growth factor, prolactin,luliberin or luteinising hormone releasing hormone, growth hormone,growth hormone releasing factor, insulin, somatostatin, glucagon,interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin,calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin,bacitracins, polymixins, colistins, tyrocidin, grarnicidines, andsynthetic analogues, modifications and pharmacologically activefragments thereof, monoclonal antibodies and soluble vaccines.

As used herein, the term “small molecule” refers to organic compounds,whether naturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Typically, small moleculeshave a molecular weight of less than about 1500 g/mol. Also, smallmolecules typically have multiple carbon-carbon bonds. Knownnaturally-occurring small molecules include, but are not limited to,penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Knownsynthetic small molecules include, but are not limited to, ampicillin,methicillin, sulfamethoxazole, and sulfonamides.

As used herein, “administering”, and similar terms means delivering thecomposition to the individual being treated. In some instances thecomposition of the invention is capable of being circulated systemicallywhere the composition binds to a target cell and is taken up byendocytosis. Thus, the composition is preferably administered to theindividual systemically, typically by subcutaneous, intramuscular,intravenous, or intraperitoneal administration. Injectables for such usecan be prepared in conventional forms, either as a liquid solution orsuspension, or in a solid form that is suitable for preparation as asolution or suspension in a liquid prior to injection, or as anemulsion. Suitable excipients include, for example, water, saline,dextrose, glycerol, ethanol, and the like; and if desired, minor amountsof auxiliary substances such as wetting or emulsifying agents, buffers,and the like can be added.

In some instances compositions, such as a polyelectrolyte, may beapplied or formed on a surface from which one or more active agents arereleased on disruption of the layers of the multilayer. The compositioncan be administered by placing the surface in contact with theenvironment to which the agent is to be released or delivered. Forexample the coated surface may be placed in contact with tissue or otherenvironment. The surface may the surface of a device, e.g., a medicaldevice, which can be implanted in tissue or in contact with skin, forexample. The type of surface is not particularly limited and may includeglass, silicon, quartz, plastic, polymer, metal or ceramic, or anybiocompatible surface.

A “pharmaceutically acceptable salt” includes a salt with an inorganicbase, organic base, inorganic acid, organic acid, or basic or acidicamino acid. As salts of inorganic bases, the invention includes, forexample, alkali metals such as sodium or potassium; alkaline earthmetals such as calcium and magnesium or aluminum; and ammonia. As saltsof organic bases, the invention includes, for example, trimethylamine,triethylamine, pyridine, picoline, ethanolamine, diethanolamine, andtriethanolamine. As salts of inorganic acids, the instant inventionincludes, for example, hydrochloric acid, hydroboric acid, nitric acid,sulfuric acid, and phosphoric acid. As salts of organic acids, theinstant invention includes, for example, formic acid, acetic acid,trifiuoroacetic acid, fumaric acid, oxalic acid, lactic acid, tartaricacid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.As salts of basic amino acids, the instant invention includes, forexample, arginine, lysine and ornithine. Acidic amino acids include, forexample, aspartic acid and glutamic acid. Anionic charge dynamicpolymers, cationic species or other agents of the compositions of thisinvention may be in the form of pharmaceutically acceptable salts.

THE EXAMPLES

General Considerations. ¹H and ¹³C nuclear magnetic resonance (NMR)spectra were recorded on Bruker AC+300 (300.135 MHz) and Varian UNITY500 (499.896 MHz) spectrometers. Chemical shift values are given in ppmand are referenced with respect to residual protons from solvent.Silicon substrates (e.g., 0.5×3.5 cm²) used for the fabrication ofmultilayered films were cleaned with methylene chloride, ethanol,methanol, and deionized water, and dried under a stream of filteredcompressed air. Surfaces were then activated by etching with oxygenplasma for 5 min (Plasma Etch, Carson City, Nev.) prior to filmdeposition. The optical thicknesses of films deposited on siliconsubstrates were determined using a Gaertner LSE ellipsometer (632.8 nm,incident angle=70°). Data were processed using the Gaertner EllipsometerMeasurement Program. Relative thicknesses were calculated assuming anaverage refractive index of 1.577 for the multilayered films.Thicknesses were determined in at least five different standardizedlocations on each substrate and are presented as an average (withstandard deviation) for each film. All films were dried under a streamof filtered compressed air prior to measurement. The pH of buffers usedfor erosion and hydrolysis experiments was recorded using a pH meterand, for the preparation of deuterated buffers, is reported as pH.Fluorescence measurements of solutions used to erode multilayered filmsfabricated from fluorescently labeled polymers were made using aFluoromax-3 fluorimeter (Jobin Yvon, Edison, N.J.). Film topography andsurface roughness were obtained from height data imaged in tapping modeon a Nanoscope Multimode atomic force microscope (Digital Instruments,Santa Barbara, Calif.), using scan rates of 10-20 μm/s to obtain 256×256pixel images. Silicon cantilevers with a spring constant of 40 N/m and aradius of curvature of <10 nm were used (model NSC15/NoAl, MikroMaschUSA, Inc., Portland, Oreg.). For each sample, at least two different 10μm×10μm scans were obtained at randomly chosen points near the center ofthe film at each time point. Height data were flattened using a2^(nd)-order fit. Root-mean squared surface roughness (R_(rms)) wascalculated over the scan area using the NanoScope® software package.

Materials. Test grade n-type silicon wafers were purchased from Si-Tech,Inc. (Topsfield, Mass.). Poly(allylamine hydrochloride) (PAH, MW≈60,000)was obtained from Alfa Aesar Organics (Ward Hill, Pa.). Citraconicanhydride and succinic anhydride were purchased from Aldrich ChemicalCo. (Milwaukee, Wis.). 7-dimethylaminocoumarin-4-acetic acid,succinimidyl ester (NHS-activated coumarin) was purchased fromInvitrogen (Carlsbad, Calif.). All other materials were used as receivedwithout further purification unless otherwise noted. Deionized water (18MΩ) was used for washing steps and to prepare all polymer solutions.Solutions of cationic polymers used for dipping (20 mM with respect tothe molecular weight of the polymer repeat unit) were prepared in 18 MΩwater and pH was adjusted to pH˜7 by using 1 N NaOH. Solutions ofanionic polymers (20 mM with respect to the molecular weight of thepolymer repeat unit) were prepared in 18 MΩ water and pH was adjusted topH˜8. All buffers and polymer solutions were filtered through a 0.2-μmmembrane syringe filter prior to use unless otherwise noted.

Synthesis of Citraconic Amide-Substituted Polymer 2. PAH (100 mg) wasdissolved in 1.0 N NaOH (3 mL) and stirred overnight. Citraconicanhydride (400 μL, ˜2 equivalents relative to amine functionality inPAH) was added dropwise to the PAH solution, and the reaction mixturewas stirred overnight at room temperature. During the reaction, aqueousNaOH (6.0 N) was added as necessary to maintain the pH of the reactionsolution above pH 8. The resulting reaction mixture was dialyzed(SpectraPor, MWCO=3500) against water (adjusted to pH>7 using NaOH) for24 hours and lyophilized to yield the final product as a white powderysolid in near quantitative yield. The addition of citraconic anhydrideto a primary amine leads to two isomers, one with a methyl groupproximal to the newly generated amide bond, and one with the methylgroup distal to the newly generated amide bond (see structure in text).The ratio of distal to proximal isomers formed under the conditionsoutlined above was determined to be ˜7:3 using ¹H NMR spectroscopy. ¹HNMR data for the final product: (D₂O) δ (ppm)=1.2 (2H, br, CH₂CHCH₂NH),1.7 (1H, br, CH₂CHCH₂NH), 1.93 (3H, s, COCHCCH₃COONa), 3.1 (2H, br,CH₂CHCH₂NH), 5.6 (proximal isomer, 0.3H, s, COCCH₃CHCOONa), 5.8 (distalisomer, 0.7H, s, COCHCCH₃COONa).

Synthesis of Succinic Amide-Substituted Polymer 3. PAH (100 mg) wasdissolved in 1.0 N NaOH (3 mL) and stirred overnight. Succinic anhydride(400 μg, ˜2 equivalents relative to amine functionality in PAH) wasadded dropwise to the PAH solution, and the reaction mixture was stirredovernight at room temperature. During the reaction, aqueous NaOH (6.0 N)was added as necessary to maintain the pH of the reaction solution abovepH 8. The resulting reaction mixture was dialyzed (SpectraPor,MWCO=3500) against water (adjusted to pH>7 using NaOH) for 24 hours andlyophilized to yield the final product as a white powdery solid in nearquantitative yield. ¹H NMR data for the final product: (D₂O) (ppm)=1.2(2H, br, CH₂CHCH₂NH), 1.7 (1H, br, CH₂CHCH₂NH), 2.5 (4H, s,COCH₂CH₂COONa), 3.1 (2H, br, CH₂CHCH₂NH).

Synthesis of Coumarin-Labeled Poly(allylamine hydrochloride). PAH (550mg) was dissolved in methanol (˜5 wt % in methanol) and 1 mL of a sodiummethoxide solution (35 wt % in methanol) was added. The resultingreaction mixture was stirred for 4 hr at 45° C., precipitated NaCl wasremoved by filtration, and NHS-activated coumarin (9.5 mg, 0.5 mol %relative to the amine repeat units in PAH) was added and the reactionmixture was stirred overnight at room temperature. One equivalent of HClwas added to the reaction mixture, and the resulting reaction productwas concentrated by rotary evaporation. The crude product was dissolvedin water and purified by dialysis (SpectraPor, MWCO=3500) againstdeionized water at ambient temperature for three days. The resultingsolution was lyophilized to yield a yellow solid that was used withoutfurther purification.

Characterization of Side-Chain Amide Hydrolysis. ¹H NMR experiments usedto characterize the kinetics of side chain hydrolysis for functionalizedpolymers were conducted in the following general manner. Polymer (˜10mg) was dissolved in either deuterated phosphate buffer (0.6 mL, 0.5 M,pH=7.4) or deuterated acetate buffer (0.6 mL, 0.5 M, pH=5).3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (˜3 mg) was addedas an internal standard, and the resulting solution was placed in aglass NMR tube. The NMR tube was kept in a 37° C. incubator and removedperiodically for analysis by ¹H NMR spectroscopy. The disappearance ofthe α-proton of the side chains at 5.6 and 5.8 ppm was monitored andintegrated versus the trimethylsilyl protons of the internal standard.

Fabrication of Multilayered Films. Multilayered films were fabricated onplanar silicon substrates using an alternate dipping procedure accordingto the following general protocol: (1) Substrates were submerged in asolution of polycation for 5 min, (2) substrates were removed andimmersed in an initial water bath for 1 min followed by a second waterbath for 1 min, (3) substrates were submerged in a solution of polyanionfor 5 min, and (4) substrates were rinsed in the manner described above.This cycle was repeated until the desired number of polycation/polyanionbilayers (typically 20) had been deposited. For experiments designed tocharacterize film growth profiles by ellipsometry, films were driedafter every five cycles of the above procedure using filtered compressedair. Films to be used in erosion and release experiments were eitherused immediately after fabrication or dried under a stream of filteredcompressed air and stored in a vacuum desiccator until use. All filmswere fabricated at ambient room temperature.

Characterization of Film Erosion and Release Kinetics. Experimentsdesigned to investigate film erosion and release kinetics were performedin the following general manner: Film-coated substrates were placed in aplastic UV-transparent cuvette, and either 1.0 mL of HEPES buffer(pH=7.4) or 1.0 mL of acetate buffer (pH=5.0) was added to covercompletely the film-coated portion of the substrates. These samples wereincubated at 37° C. and removed at predetermined intervals forcharacterization by ellipsometry. Films were rinsed under deionizedwater and dried under a stream of filtered compressed air prior tomeasurement. Values of optical film thickness were determined in atleast five different predetermined locations on the substrate byellipsometry and the samples were returned immediately to the buffersolution. For experiments designed to monitor the concentrations offluorescently labeled PAH released into solution, fluorescencemeasurements were made using the solution used to incubate the sample(excitation wavelength=376 nm; emission wavelength=470 nm).

Citraconic amide-functionalized polymer 2 was synthesized by thering-opening addition of citraconic anhydride to PAH in analogy tomethods described previously for the functionalization of small-moleculeamines and polyamines.³⁰⁻³⁶ Treatment of PAH with an excess ofcitraconic anhydride in an aqueous NaOH solution at room temperatureresulted in exhaustive functionalization with citranonicamide-functionalized side chains, as determined by ¹H NMR spectroscopy(Eq 3). The addition of citraconic anhydride to a primary amine can leadto the generation of two isomers, one with a methyl group distal to thenewly generated amide bond, and one with the methyl group proximal tothe newly generated amide bond (e.g., see Eq 3). The ratio of distal toproximal side chain isomers in

samples of polymer 2 used in this study was determined to be ˜7:3 byintegration of the α-protons of the side chains using ¹H NMRspectroscopy. Polymer 2 was isolated as a solid in near quantitativeyield and was soluble in aqueous buffers at all concentrations requiredfor subsequent experiments described below.

As described above, past studies report that citraconic amides arerelatively stable in alkaline media, but they hydrolyze readily inacidic media.30-35 Characterization of the kinetics of hydrolysis of theside chains of polymer 2 upon incubation in deuterated phosphate buffer(pH=7.4) at 37° C. using 1H NMR spectroscopy demonstrated that sidechain hydrolysis occurred slowly at near-neutral pH. As shown in FIG. 2(closed squares), only ˜25% of the side chains of polymer 2 werehydrolyzed after incubation in phosphate buffer for eight days. However,the side chains of polymer 2 were hydrolyzed rapidly when the polymerwas incubated at lower pH. For example, as shown in FIG. 2 (closedtriangles), ˜55% of side chains were hydrolyzed within the first fivehours when polymer 2 was incubated in deuterated acetate buffer(pH=5.0). These pH-dependent results are consistent with the results ofpast studies.30-35

The addition of citraconic anhydride to poly(allylamine) results in theformation of two side chain isomers (see Eq 2). Further inspection ofthe data in FIG. 2 reveals that the rate of hydrolysis of the sidechains of polymer 2 that have the methyl group distal to the amide bondis significantly greater than the rate of hydrolysis of the side chainshaving the methyl group proximal to the amide bond (FIG. 2, dashedlines; individual data for hydrolysis at pH 7.4 not shown). Hydrolysisof the distal isomer occurs very rapidly (e.g., ˜70% conversion; opendiamonds) within the first five hours and is >95% complete after 70hours. By contrast, hydrolysis of the proximal isomer occurs much moreslowly, and is only ˜20% complete after incubation for eight days. Theselarge differences in rate are consistent with the results of paststudies reporting the significant influence of substituent effects onthe hydrolysis of amides formed using α-methyl derivatives of maleicanhydride. In the context of this current investigation, these results,when combined, demonstrate that polymer 2 is converted over a period ofseveral days to a polymer that contains a majority of side chains thatare cationic (i.e., primary amines, with the remainder being citraconicamide proximal isomers that continue to hydrolyze more slowly) uponincubation in acidic media. In general, the actual net charge of polymer2 would depend upon a number of different factors, including the extentof side chain hydrolysis as well as environmental factors such assolution pH and ionic strength (the relative net charges and extents ofreaction depicted in FIG. 1 are indicated for illustrative purposesonly).

Fabrication and Characterization of Polyelectrolyte MultilayersFabricated Using Polymer 2.

A series of experiments was performed to determine whether polymer 2could be used to fabricate polyelectrolyte multilayers using PAH and analternate dipping procedure similar to that used in numerous paststudies.¹⁻⁴ For these and all subsequent experiments described below,multilayered films were fabricated layer-by-layer directly on thesurfaces of planar silicon substrates to facilitate the characterizationof film growth and increases or decreases in film thickness usingellipsometry.

FIG. 3 shows a plot of optical film thickness versus the number ofPAH/polymer 2 layer pairs (referred to hereafter as ‘bilayers’)deposited. Inspection of these data (filled squares) reveals an averagefinal film thickness of ˜90 nm after the deposition of 20 bilayers. Filmthickness increased in a manner that was supra-linear, rather thanlinear, with respect to the number of bilayers deposited. This filmgrowth behavior is similar to that reported recently for the‘exponential’ growth of polyelectrolyte multilayers fabricated from avariety of different weak polycation/polyanion pairs,⁴⁰⁻⁴⁵ and couldresult from the ability of one or both polyelectrolytes to diffusewithin these films during assembly.⁴⁰ Characterization of the surface ofa film 20 bilayers thick using atomic force microscopy revealed thesefilms to be continuous with a root-mean squared (R_(rms)) surfaceroughness of ˜13 nm.

Time-dependent hydrolysis of the amide functionality in the side chainsof anionic polymers of this invention, particularly polymer 2 [which,ultimately, results in the conversion of the anionic polymer to acationic poly(amine)] can be used to (i) change the nature of theelectrostatic interactions in multilayered assemblies, and (ii) promotefilm disruption and disassembly in ways that can be used to providecontrol over the release of cationic film components.

To demonstrate this, a series of experiments were performed using filmsfabricated from polymer 2 and PAH labeled with a fluorescent coumarinderivative (PAH_(FL)). PAH_(FL) was used in these experiments tofacilitate characterization of the time-dependent release of PAH fromfilms incubated in aqueous environments (described below). The growthprofiles of films fabricated using polymer 2 and PAH_(FL) did not varysignificantly from the growth profile shown in FIG. 3 using polymer 2and unlabeled PAH (as determined by ellipsometry, data not shown).

The stability (or instability) of films fabricated from polymer 2 andPAH_(FL) in near-neutral or acidic media was investigated by incubatingthese assemblies in HEPES buffer (pH=7.4) or acetate buffer (pH=5.0) at37° C. FIG. 4 shows a plot of decreases in optical film thickness as afunction of time measured during the incubation of films fabricated from20 bilayers of polymer 2 and PAH_(FL) (˜90 nm thick). Inspection ofthese data reveals these films to be stable (that is, they do notdecrease significantly in optical thickness) upon incubation at pH 7.4for approximately seven days (closed squares), but that these filmsdecrease in thickness completely, and with a profile that is essentiallylinear, over a period of 48 hours when incubated at pH 5.0 (opensquares).

The large, pH-dependent differences in the stability and erosionprofiles of the PAH_(FL)/polymer 2 films shown in FIG. 4 are consistentwith the large and pH-dependent differences in the relative rates ofsolution-phase amide side chain hydrolysis shown in FIG. 2. Theseobservations, when combined, provide general support for the view thatpolymer 2 destabilizez multilayers and promotes film disassembly througha time-dependent ‘charge-shifting’ mechanism that involves polymerside-chain hydrolysis.

The erosion of these materials at pH 5.0 could also arise from otherfactors, such as changes in the percent ionization of polymer 2 thatcould occur when these films are incubated in acidic environments.Several past studies have demonstrated, for example, thatpolyelectrolyte multilayers fabricated from weak polyacids [e.g.,poly(acrylic acid), poly(methacrylic acid), etc.] can be dissolved and‘erased’⁴⁶⁻⁵⁰ or transformed physically in other ways^(51,52) whenexposed to changes in pH that change the ionic character of the polymersand disrupt interpolyelectrolyte interactions (e.g., by protonation ordeprotonation of anionic carboxylate functionality). In general, pastreports describing film dissolution in multilayers fabricated using weakpolyelectrolytes describe transformations that often occur very rapidly(e.g., on the order of minutes),^(46,49,52) rather than over the periodof ˜2 days as observed in our experiments.

To provide additional support for the charge-shifting hypothesis, anadditional series of experiments were conducted using films fabricatedfrom PAH and succinic amide-functionalized polymer 3. Polymer 3 is ananalog of polymer 2 synthesized by the ring-opening addition of succinicanhydride to PAH (Eq 4). Polymer 3 is particularly useful as a controlin these experiments for several reasons: (i) because succinic anhydridedoes not contain the double bond functionality present in citraconicanhydride, succinic amides are more conformationally flexible thancitraconic amides, (ii) the terminal carboxylate functionality ofsuccinic amides is thus not maintained in close proximity to the amidegroup, and, as a result (iii) rates of hydrolysis of succinic amides aremuch slower than the corresponding rates of hydrolysis of citraconicamides.³³

FIG. 3 (dashed line) shows a plot of film thickness for a PAH/polymer 3film versus the number of bilayers of polymer 3 and PAH deposited, anddemonstrates that the conformational flexibility of the polymer sidechain does not influence film growth significantly relative to filmsfabricated from polymer 2. However, striking differences in thestability of films fabricated from polymer 3 were observed when thesefilms were incubated in aqueous media. Inspection of the data in FIG. 3(dashed lines) reveals that films fabricated from polymer 3 are stableand do not decrease in optical thickness for up to seven days uponincubation at either pH 7.4 or pH 5.0. These results provide additionalstrong support for the view that the erosion of films fabricated frompolymer 2 occurs as a result of the hydrolysis of the side chains inpolymer 2 (and a concomitant change in the net charge of the polymer),and not as a result of other factors (such as changes in pH, ionicstrength, or the percent ionization of the polymer) that could occurduring the incubation of these films in acidic environments.

The gradual erosion of films fabricated from polymer 2 and PAH_(FL) inacidic environments results in the gradual and controlled release ofPAH_(FL) into solution. FIG. 4 shows a plot of solution fluorescenceintensity versus time measured during the film incubation and erosionexperiments described above in FIG. 3. Inspection of these data revealsthat PAH_(FL) was released gradually into solution for up to ˜100 hours(open squares) when films fabricated from polymer 2 were incubated at pH5.0. By contrast, levels of solution fluorescence observed during theincubation of these films at pH 7.4 (closed squares) were very low overthe entire course of the experiment.

These data are consistent with the pH-dependent results of film erosiondiscussed above and demonstrate that polymer 2 can be used to designpolyelectrolyte multilayers that are stable at neutral or near-neutralpH, but permit control over the surface-mediated release of a cationicagent for approximately four days under acidic conditions. On the basisof these solution fluorescence measurements, it is estimated that films20 bilayers thick contained approximately 20 μg of PAH_(FL) per cm².Because layer-by-layer assembly can be used to exert precise controlover film thickness and, thus, the amount of PAH incorporated (e.g., bycontrolling the number of layers of polymer deposited), this generalapproach can be used to increase (or decrease) the amounts of PAH orother functional cationic agents incorporated into and released fromthese materials. Finally, the remaining data in FIG. 5(dashed lines)correspond to cumulative amounts of solution fluorescence measuredduring the incubation of control films fabricated from PAH_(FL) andpolymer 3. These data demonstrate that these films do not releasesignificant amounts of PAH_(FL) and are consistent with the results ofthe erosion experiments described above.

The use of ‘charge-shifting’ anionic polymers provides control over thedisruption of ultrathin multilayered polyelectrolyte films in aqueousenvironments. The addition of certain anhydrides to poly(alkeneamine),and particularly the use of citraconic anhydride to poly(allylamine), acommercially-available polyamine used widely to fabricatepolyelectrolyte multilayers, yields an anionic,carboxylate-functionalized polymer (e.g., polymer 2) that can beconverted readily back to cationic poly(alkeneamine) in acidicenvironments (e.g., pH 5). The incorporation of such polymers, e.g.,polymer 2, as an anionic component in polyelectrolyte multilayersprovides an approach to the fabrication of films that are relativelystable at neutral pH (e.g., pH ˜7) but that erode over a period of time,in some cases over several days, when exposed to low pH environments(e.g., pH ˜5). Control experiments using a structural analog of polymer2 functionalized with carboxylate side chains that do not hydrolyze asreadily as those of polymer 2 provided support that the disruption ofthese films occurred as a result of polymer side chain hydrolysis (and aresulting change in the net charge of the polymer) and not as a resultof other factors, such as changes in pH or ionic strength, that couldoccur upon the incubation of these assemblies. These results alsoindicate that the rate of disruption of such multilayer films can becontrolled by selection of the amide groups attached to the polymerbackbone. For example, the relative amounts of amide side chains havingdouble bonds and those not having double bonds can be varied to vary therate and extent of side chain hydrolysis and the rate and extent of filmdisruption.

Because the method of this invention is based upon the use of anionicpolymers to induce film instability, it provides a platform for thedesign of polyelectrolyte multilayers that can be used to providecontrol over the release of cationic film components. In a specificembodiment, ultrathin films (e.g., ˜100 nm thick) fabricated usingpolymer 2 sustain the release of fluorescently labeled PAH for up tofour days when incubated at pH 5.0.

The work described here differs fundamentally from approaches that havebeen reported previously for the disruption of polyelectrolytemultilayers using degradable cationic polymers, significantly expandsthe range of different cationic agents (e.g., cationic proteins,peptides, polymers, nanoparticles, etc.) that can be released ordelivered from surfaces using polyelectrolyte multilayers.

In addition, the synthetic approach used here is modular and can be usedto introduce anionic ‘charge-shifting’ character to a broad range ofother primary amine-functionalized polymers. The method of thisinvention can be used to tune the ‘charge-shifting’ character of thesepolymers over a broad range of times, and for a broad range of potentialapplications, by varying either the numbers or the structures of the‘charge-shifting’ anionic side chains incorporated. The relativeinstability of these materials at pH values representative of thosefound in the endosomes and lysosomes of cells also suggestsopportunities to design film-coated particles or hollow multilayermicrocapsules designed for the intracellular delivery of therapeuticmacromolecules.⁵³

The present methods may be carried out by performing any of the stepsdescribed herein, either alone or in various combinations. The presentcompounds may also have any or all of the components described herein.One skilled in the art will recognize that all embodiments of thepresent invention are capable of use with all other embodiments of theinvention described herein. Additionally, one skilled in the art willrealize that the present invention also encompasses variations of thepresent methods and compositions that specifically exclude one or moreof the steps, components or groups described herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

Accordingly, for all purposes, the present invention encompasses notonly the main group, but also the main group absent one or more of thegroup members. The present invention also envisages the explicitexclusion of one or more of any of the group members in the claimedinvention.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. A number of specific groups of variabledefinitions have been described herein. It is intended that allcombinations and subcombinations of the specific groups of variabledefinitions are individually included in this disclosure. Accordingly,for all purposes, the present invention encompasses not only the maingroup, but also the main group absent one or more of the group members.The present invention also envisages the explicit exclusion of one ormore of any of the group members in the claimed invention.

Compounds described herein may exist in one or more isomeric forms,e.g., structural or optical isomers. When a compound is described hereinsuch that a particular isomer, enantiomer or diastereomer of thecompound is not specified, for example, in a formula or in a chemicalname, that description is intended to include each isomers andenantiomer (e.g., cis/trans isomers, R/S enantiomers) of the compounddescribed individual or in any combination. Additionally, unlessotherwise specified, all isotopic variants of compounds disclosed hereinare intended to be encompassed by the disclosure. For example, it willbe understood that any one or more hydrogens in a molecule disclosed canbe replaced with deuterium or tritium. Isotopic variants of a moleculeare generally useful as standards in assays for the molecule and inchemical and biological research related to the molecule or its use.Isotopic variants, including those carrying radioisotopes, may also beuseful in diagnostic assays and in therapeutics. Methods for making suchisotopic variants are known in the art. Specific names of compounds areintended to be exemplary, as it is known that one of ordinary skill inthe art can name the same compounds differently.

Molecules disclosed herein may contain one or more ionizable groups[groups from which a proton can be removed (e.g., —COOH) or added (e.g.,amines) or which can be quaternized (e.g., amines)]. All possible ionicforms of such molecules and salts thereof are intended to be includedindividually in the disclosure herein. With regard to salts of thecompounds herein, one of ordinary skill in the art can select from amonga wide variety of available counterions those that are appropriate forpreparation of salts of this invention for a given application. Inspecific applications, the selection of a given anion or cation forpreparation of a salt may result in increased or decreased solubility ofthat salt. Every formulation or combination of components described orexemplified herein can be used to practice the invention, unlessotherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. The broad termcomprising is intended to encompass the narrower consisting essentiallyof and the even narrower consisting of. Thus, in any recitation hereinof a phrase “comprising one or more claim element” (e.g., “comprising Aand B), the phrase is intended to encompass the narrower, for example,“consisting essentially of A and B” and “consisting of A and B.” Thus,the broader word “comprising” is intended to provide specific support ineach use herein for either “consisting essentially of” or “consistingof.” The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, catalysts, reagents, synthetic methods, purification methods,analytical methods, and assay methods, other than those specificallyexemplified can be employed in the practice of the invention withoutresort to undue experimentation. All art-known functional equivalents,of any such materials and methods are intended to be included in thisinvention. The terms and expressions which have been employed are usedas terms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed byexamples, preferred embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

All references cited herein are hereby incorporated by reference to theextent that there is no inconsistency with the disclosure of thisspecification. Some references provided herein are incorporated byreference to provide details concerning sources of starting materials;alternative starting materials, reagents, methods of synthesis,purification methods, and methods of analysis; as well as additionaluses of the invention.

Unless otherwise specified, “a” or “an” means “one or more”.

While certain specific embodiments have been illustrated and described,it should be understood that changes and modifications can be madetherein in accordance with ordinary skill in the art without departingfrom the invention in its broader aspects as defined in the followingclaims.

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1. A method for controlled delivery of a neutral, zwitterionic, anionic,or cationic agent to a selected environment which comprises the stepsof: forming a polyelectrolyte multilayer comprising one or more cations,an anionic polymer and a neutral, zwitterionic, anionic, or cationicagent, where the polyelectrolyte multilayer is in contact with theselected environment; and selectively removing one or more functionalgroups from the anionic polymer, thereby decreasing the anionic chargeof the polymer, disrupting at least one layer of the multilayer andreleasing the one or more cations and the neutral, zwitterionic,anionic, or cationic agent into the selected environment; wherein theanionic polymer comprises a polymeric backbone formed from repeat units,and one or more removable functional groups attached to the polymericbackbone and distributed throughout the anionic polymer, wherein theanionic polymer has an anionic charge density which decreases when oneor more of the removable functional groups is removed from the dynamiccharge state anionic polymer and wherein the anionic polymer is apolymer other than poly(lysine).
 2. The method of claim 1 wherein theselected environment is human or animal tissue.
 3. The method of claim 1wherein the one or more removable functional groups are anionic.
 4. Themethod of claim 1 wherein the one or more removable anionic functionalgroups are linked to the polymer backbone by an amide linkage.
 5. Amethod for controlled delivery of a neutral, zwitterionic, anionic, orcationic agent to a selected environment which comprises the steps of:forming a polyelectrolyte multilayer comprising one or more cations, ananionic polymer and a neutral, zwitterionic, anionic, or cationic agent,where the polyelectrolyte multilayer is in contact with the selectedenvironment; and selectively removing one or more functional groups fromthe anionic polymer, thereby decreasing the anionic charge of thepolymer, disrupting at least one layer of the multilayer and releasingthe one or more cations and the neutral, zwitterionic, anionic, orcationic agent into the selected environment; wherein the anionicpolymer comprises a polymeric backbone formed from repeat units, and oneor more removable functional groups attached to the polymeric backbone,and wherein the anionic polymer has an anionic charge density whichdecreases when one or more of the removable functional groups is removedfrom the dynamic charge state anionic polymer, wherein the anionicpolymer has the formula:

where r is an integer ranging from 5 to 100,000, x and y are numbersrepresenting the mole percent of the indicated side chains in thepolymer where x+y is 1 and y represents from 10 to 100 mole percent ofamide side chain, wherein x can be zero, the wavy line represents alinker which covalently attaches the primary amine or the amide to thepolymer backbone, n is 0 or 1, dotted lines indicate optional bonds, ifR₃ and R₄ are absent the bond in the ring is a double bond; and whereR₁, R₂, and R₃ and R₄ are selected from optionally substituted alkyl,alkenyl, alkynyl, aryl, heteroaryl, carbocyclic and heterocyclic groupsand wherein R₁ and R₂ can together form an optionally substituted 5-10member carbocyclic or heterocyclic ring which may be aryl or heteroarylor which can contain one or two double bonds, where optionalsubstituents include one or more halogen, hydroxyl, alkyl, alkenyl,alkynyl, aryl, heteroaryl, carbocyclic or heterocyclic groups —CN,alkoxyl, —COOH (or —COO⁻), —COOR, or —CON(R′)₂, where R is alkyl,alkenyl, alkynyl, aryl, heteroaryl, carbocyclic or heterocyclic and eachR′ is hydrogen or R, and wherein the anionic polymer contains two ormore different amide side chains, where any of the variables R₁-R₄ inthe different amide side chains are different, n is different or theoptionally double bond in the amide is present or absent.
 6. The methodof claim 5 wherein the linker to the polymer backbone is an alkylene orallkyleneoxy linker.
 7. The method of claim 5 wherein the anionicpolymer has two different amide side chains.
 8. The method of claim 7wherein the two different amide side chains of the anionic polymer arestereoisomers.
 9. The method of claim 5 wherein x is zero in the anionicpolymer.
 10. A method for controlled delivery of a neutral,zwitterionic, anionic, or cationic agent to a selected environment whichcomprises the steps of: forming a polyelectrolyte multilayer comprisingone or more cations, an anionic polymer and a neutral, zwitterionic,anionic, or cationic agent, where the polyelectrolyte multilayer is incontact with the selected environment; and selectively removing one ormore functional groups from the anionic polymer, thereby decreasing theanionic charge of the polymer, disrupting at least one layer of themultilayer and releasing the one or more cations and the neutral,zwitterionic, anionic, or cationic agent into the selected environment;wherein the anionic polymer comprises a polymeric backbone formed fromrepeat units, and one or more removable functional groups attached tothe polymeric backbone, and wherein the anionic polymer has an anioniccharge density which decreases when one or more of the removablefunctional groups is removed from the dynamic charge state anionicpolymer; wherein the anionic polymer has the formula:

where r is an integer ranging from 5 to 100,000, m is an integer rangingfrom 1 to 10, y¹ and y² are each integers representing the mole percentof the indicated side chains compared to the total of y¹+y² in thepolymer and R₁ is selected from optionally substituted alkyl, alkenyl,alkynyl, aryl, heteroaryl, carbocyclic and heterocyclic where optionalsubstituents include one or more halogen, hydroxyl, alkyl, alkenyl,alkynyl, aryl, heteroaryl, carbocyclic or heterocyclic groups, —CN,alkoxyl, —COOH (or —COO⁻), —COOR, or —CON(R′)₂, where R is alkyl,alkenyl, alkynyl, aryl, heteroaryl, carbocyclic or heterocyclic and eachR′ is hydrogen or R.
 11. The method of claim 10 wherein in the anionicpolymer R₁ is an alkyl group having 1 to 3 carbon atoms.
 12. The methodof claim 10 wherein in the anionic polymer R₁ is an alkyl group having 1to 3 carbon atoms and m is 1, 2 or
 3. 13. The method of claim 1 whereinthe anionic polymer is a copolymer.
 14. The method of claim 1 whereinthe anionic polymer is a block copolymer wherein at least one blockcomprises a polymeric backbone formed from repeat units, and one or moreremovable functional groups attached to the polymeric backbone.
 15. Themethod of claim 10 wherein the anionic polymer has the formula:

where r is an integer ranging from 5 to 100,000, m is 1-6, y¹ and y² arenumbers representing the mole percent of the indicated side group wherey¹+y² is 1; and R₁ is an optionally substituted C1-C6 alkyl group. 16.The method of claim 15 wherein y¹ ranges from 0.1 to 0.9 in the anionicpolymer.
 17. The method of claim 1 wherein the anionic polymer has theformula:

where r is an integer ranging from 5 to 100,000, m is 1-6, y¹, y² and y³are numbers representing the mole percent of the indicated side groupwhere y¹+y²+y³ is 1; and R₁ is an optionally substituted C1-C6 alkylgroup.
 18. The method of claim 17, wherein y³ ranges from 0.01 to 0.90in the anionic polymer.
 19. The method of claim 17 wherein y³ rangesfrom 0.25 to 0.50 in the anionic polymer.
 20. The method of claim 1wherein the one or more agents are a protein, peptide, a small molecule,a natural polymer or a synthetic polymer.
 21. The method of claim 1wherein the one or more agents are nanoparticles.
 22. The method ofclaim 1 wherein the one or more agents are therapeutic, diagnostic orprophylactic agents.
 23. The method of claim 1 wherein the one or moreagents is a cationic agent.
 24. The method of claim 1 wherein the one ormore agents is a neutral, zwitterionic or anionic agent.
 25. A methodfor delivery of one or more cationic or anionic agents to a cell ortissue which comprises the steps of: forming an interpolyelectrolytecomplex between anionic species and cationic species wherein the anionicspecies includes an anionic polymer and the anionic species or cationicspecies includes one or more anionic or cationic agents, wherein theanionic polymer comprises a polymeric backbone formed from repeat units,and one or more removable functional groups attached to the polymericbackbone and distributed throughout the anionic polymer, wherein theanionic polymer has an anionic charge density which decreases when oneor more of the removable functional groups is removed from the dynamiccharge state anionic polymer and wherein the anionic polymer is apolymer other than poly(lysine); contacting the cell or tissue with theinterpolyelectrolyte complex; and removing one or more functional groupsfrom the anionic polymer to release the one or more anionic or cationicagents.
 26. A method for delivery of one or more cationic or anionicagents to a cell or tissue which comprises the steps of: forming aninterpolyelectrolyte complex between anionic species and cationicspecies wherein the anionic species includes an anionic polymer and theanionic species or cationic species includes one or more anionic orcationic agents, wherein the anionic polymer comprises a polymericbackbone formed from repeat units, and one or more removable functionalgroups attached to the polymeric backbone, wherein anionic charge isdistributed throughout the anionic polymer, wherein the anionic polymerhas an anionic charge density which decreases when one or more of theremovable functional groups is removed from the dynamic charge stateanionic polymer; contacting the cell or tissue with theinterpolyelectrolyte complex; and removing one or more functional groupsfrom the anionic polymer to release the one or more anionic or cationicagent wherein the anionic polymer has the formula:

where r is an integer ranging from 5 to 100,000, m is 1-6, y¹ and y² arenumbers representing the mole percent of the indicated side group wherey¹+y² is 1; and R₁ is an optionally substituted C1-C6 alkyl group. 27.The method of claim 1 wherein the polyelectrolyte multilayer comprisesmore than one layer.
 28. The method of claims 1 wherein thepolyelectrolyte multilayer is formed from one or more cationic polymersand the anionic polymer.
 29. The method of claim 5 wherein the anionicpolymer has the formula:

wherein any of the variables R₁-R₄ in the different amide side chainsare different, n is different or the optionally double bond in the amideis present or absent.